Novel neutralizing immunogen (nimiv) of rhinovirus and its uses for vaccine applications

ABSTRACT

The invention relates to methods and compositions for preventing or treating human rhinovirus infection.

FIELD OF THE INVENTION

The invention relates to methods and compositions for preventing ortreating human rhinovirus infection.

BACKGROUND OF THE INVENTION

Human rhinoviruses (HRVs) represent the single most importantetiological agents of the common cold (Arruda et al., J. Clin.Microbiol. 35:2864-2868 (1997); Couch, “Rhinoviruses.” In: Fields, B.N., Knipe, D. M. (Eds.), Virology. Raven Press, New York, 607-629(1990); Turner, Antivir. Res. 49(1):1-14 (2001)). HRVs causing aboutone-third of the outbreaks of the common cold are represented by about100 serotypes, the convalescent sera from patients infected with whichare not fully cross-neutralizing. Although HRV-induced upper respiratoryillness is often mild and self-limiting, the socioeconomic impact causedby missed work or school is enormous and the degree of inappropriateantibiotic use is significant. It has been estimated that upperrespiratory disease accounts for at least 25 million absences from workand 23 million absences of school annually in the United States (Anzuetoet al., Chest 123(5):1664-1672 (2003); Rotbart, Antivir. Res. 53:83-98(2002)).

There is increasing evidence of a link between HRV infection and moreserious medical complications. For example, HRV-induced colds are theimportant predisposing factors to acute otitis media and sinusitis, andare major factors in the induction of exacerbations of asthma in adultsand children. HRV infections are also associated with lower respiratorytract syndromes in individuals with cystic fibrosis, bronchitis, andother underlying respiratory disorders (Gern, Pediatr. Infect. Dis. J.23:S78-S86 (2004); Anzueto et al., Chest 123(5):1664-1672 (2003); Gem etal., Clin. Microbiol. Rev. 12(1):9-18 (1999); Pitkaranta et al., J.Clin. Microbiol. 35:1791-1793 (1997); Pitkaranta et al., Pediatrics102:291-295 (1998); Rotbart, Antivir. Res. 53:83-98 (2002)).

To date, no effective antiviral therapies have been approved for eitherthe prevention or treatment of diseases caused by HRV infection. Thus,there exists a significant unmet medical need to find agents that canprevent HRV infection, shorten the duration of HRV-induced illness,lessen the severity of symptoms, minimize secondary bacterial infectionsand exacerbations of underlying disease, and reduce virus transmission.A prophylactic HRV vaccine should be protective against a wide varietyof serotypes to reduce the number of HRV infections and their clinicalimpact.

Attempts to make HRV vaccines based on synthetic peptides correspondingto conserved regions of structural proteins alone (McCray et al., Nature329:736-738 (1987)) or as a part of biological fusions (Brown et al.,Vaccine 9:595-601 (1991); Francis et al., Proc. Natl. Acad. Sci. U.S.A.87:2545-2549 (1990)) have had limited success, due to low immunogenicityof chosen peptides, which may be partially explained by their lowexposure on the virus surface (limited access to antibodies) orconformational constraints.

The present invention overcomes these limitations and features a vaccinethat elicits a protective serotype cross-reactive neutralizing antibodyresponse to prevent and treat HRV infection.

SUMMARY OF THE INVENTION

The invention provides isolated rhinovirus neutralizing immunogen IV(NimIV) peptides. These peptides can be from any serotype of rhinovirus,such as human rhinoviruses (e.g., HRV14). The peptides can include, forexample, amino acids 277-283 (e.g., amino acids 275-285) of the carboxylterminal region of virus structural protein 1 (VP 1) of a humanrhinovirus. Exemplary sequences include the following: PVIKKR, PVIKKRK(HRV14), PVIKKRE (HRV6 and HRV72), PVIKKRS(HRV92), PVIEKRT (HRV83),PKIIKKR (HRV86), PVIKRRE (HRV35), PIIAKRE (HRV79), TIIKKRT (HRV3),NTEPVIKKRKGDIKSY (HRV14), and A-X₁-X₂-I-X₃-X₄-R-X₅-B, where X₁=P or T;X₂=V, K, or I; X₃=K, E, I, or A; X₄=K or R; X₅=S, E, D, T, R, T, or K;A=0-10 additional amino acids; and B=0-10 additional amino acids.

The invention also includes isolated nucleic acid molecules encoding aNimIV peptides or complements thereof. Further, the invention includesvectors (e.g., HRV14 vectors) including the peptides and nucleic acidmolecules of the invention. The vectors can be, for example, humanrhinovirus vectors, e.g., human rhinovirus vectors of a serotypedifferent from that of the human rhinovirus from which the NimIV peptideis derived. In one example, the NimIV peptide or nucleic acid moleculeis present in said human rhinovirus vector in place of NimIV sequencesoriginally present in said vector. In other examples, the humanrhinovirus from which the NimIV peptide is derived is human rhinovirus 6(HRV6) or human rhinovirus 72 (HRV72). The latter peptides may beincluded in, e.g., a human rhinovirus 14 (HRV14) vector. In otherexamples, the VP 1 protein or nucleic acid molecule of the vector isreplaced with the VP1 protein or nucleic acid of the human rhinovirusfrom which the NimIV peptide is derived. In additional examples, thevector includes an inactivated human rhinovirus, to which the NimIVpeptide is cross-linked, or a hepatitis B core sequence to which NimIVsequences are fused (see, e.g., Fiers et al., Virus Res. 103:173-176,2004; WO 2005/055957; US 2003/0138769 A1; US 2004/0146524A1; US2007/0036826 A1).

The invention further includes pharmaceutical compositions including thepeptides, nucleic acid molecules, and vectors described herein.Optionally, the pharmaceutical compositions also include one or more ofa pharmaceutically acceptable diluents, excipients, carriers, and/oradjuvants. Exemplary adjuvants include chitin microparticles andaluminum compounds. Further, the compositions can optionally include oneor more additional human rhinovirus neutralizing immunogens.

Also included in the invention are methods of inducing an immuneresponse to a rhinovirus in a subject. These methods involveadministering to the subject an isolated NimIV peptide or nucleic acidmolecule. In some examples, the subjects does not have but is at risk ofdeveloping rhinovirus infection. In other examples, the subject hasrhinovirus infection.

DEFINITIONS

By “administration” or “administering” is meant a method of giving adosage of a composition of the invention to a mammal (e.g., a human),where the method is, e.g., intranasal, topical, systemic, inhalation,oral, intravenous, sub-cutaneous, intravascular, intra-arterial,intratumor, intraperitoneal, intraventricular, intraepidural, nasal,rectal intrascleral, ophthalmic, intraocular, or intramuscular. Thepreferred method of administration can vary depending on variousfactors, e.g., the components of the pharmaceutical composition, site ofthe potential or actual disease (e.g., the location of a tumor orvascular condition to be treated) and the severity of disease.

By “human rhinovirus” (HRV) is meant any member of the familyPicornaviridae genus Rhinovirus. HRV can be classified by serotype, ofwhich approximately 100 are known to exist. For example, HRV14, HRV6,HRV37, and HRV92 refer to human rhinoviruses of serotypes number 14, 6,37, and 92 respectively.

By “pharmaceutically acceptable carrier” is meant a carrier that isphysiologically acceptable to a treated mammal, while retaining theprophylactic or therapeutic properties of the compound with which it isadministered. One exemplary pharmaceutically acceptable carrier isphysiological saline. Other physiologically acceptable carriers andtheir formulations are known to those skilled in the art and examplesare described, for example, in Remington's Pharmaceutical Sciences,(18^(th) edition), ed. A. Gennaro, 1990, Mack Publishing Company,Easton, Pa. incorporated herein by reference.

By “neutralizing immunogen” (Nim) is meant a human rhinovirus (HRV)sequence that, upon introduction into a human, elicits anti-HRVneutralizing antibodies. In the case of recombinant HRV vaccines asdescribed herein, the NimIV serotype is placed in superscript tospecifically describe the source of the Nim (e.g., NimIV^(HRV6) refersto a NimIV sequence derived from the HRV6 serotype).

A “neutralizing immunogen IV peptide” or “NimIV peptide” is a peptidehaving a sequence from the carboxyl terminal region (e.g., amino acids274-289, using HRV14 (NTEPVIKKRKGDIKSY) as a reference; see FIG. 12B) ofa rhinovirus virus structural protein 1 (VP1). NimIV peptides caninclude the specified sequences, additional flanking sequences, or onlya core, conserved sequence, as described below. In addition, thepeptides may be unmodified, and thus be identical to naturally occurringNimIV sequences, or may include one or more substitutions, deletions,insertions, or other modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25substitutions, deletions, or insertions), provided that immunogenicityof the peptide is substantially maintained. Further, the NimIV peptidesmay comprise L or D amino acids, or mixtures thereof.

Examples of NimIV peptide sequences that can be used in the inventionare listed below. The peptides can be, for example, 5-30, 8-25, 10-20,14-19, 15-18, or 16-17 amino acids in length. The peptides may include acore NimIV sequence and, optionally, be flanked with additional NimIVsequences or linker sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids on amino and/or carboxyl terminal ends).

Examples of core NimIV sequences include PVIKKR, PVIKKRK (HRV14),PVIKKRE (HRV6 and HRV72), PVIKKRS (HRV92), PVIEKRT (HRV83), PKIIKKR(HRV86), PVIKKRE (HRV35), PIIAKRE (HRV79), TIIKKRT (HRV3), TIVKKRT(HRV3), TAIVTRP (HRV2), VAIRPRT (HRV16), TAIVRRN (HRV1A),NTEPVIKKRKGDIKSY (HRV14), as well as other HRV sequences that align withthese sequences (see, for example, FIG. 11). The core sequence may bedefined, for example, by the formula A-X₁-X₂-I-X₃-X₄-R-X₅-B, where X₁=Por T; X₂=V, K, or I; X₃=K, E, I, or A; X₄=K or R; X₅=S, E, D, T, R, T,or K; A=0-10 additional amino acids; and B=0-10 additional amino acids.The sequence of A and/or B can be naturally occurring NimIV/VP1sequences, artificial sequences (e.g., linker sequences), or mixturesthereof.

A “neutralizing immunogen IV nucleic acid molecule” or “NimIV nucleicacid molecule” is a nucleic acid molecule encoding a NimIV peptide asdefined herein or the complement thereof.

A NimIV peptide or nucleic acid molecule is “isolated” if it does notinclude flanking sequences with which it is contiguous in naturallyoccurring virus. Such peptides or nucleic acid molecules may be limitedby, for example, the full-length sequence of VP1, the carboxyl terminalhalf of VP1, the carboxyl terminal quarter of VP1, or the carboxylterminal 15-30 amino acids of VP1, or corresponding regions of nucleicacid sequences (see, e.g., Laine et al., J. Gen. Virol. 87:129-138,2006).

A NimIV peptide “consists essentially of” a specified sequence, if itincludes only that sequence, as well as possibly a minimal amount offlanking sequences (e.g., 1-10, 2-9, 3-8, 4-7, or 5-6 amino acids), onamino and/or carboxyl terminal ends, which may be naturally occurringsequences, artificial sequences (e.g., linkers), or combinationsthereof. Such sequences can be present in the context of largersequences (e.g., heterologous virus or other vector sequences).

A NimIV nucleic acid molecule “consists essentially of” a specifiedsequence, if it includes only that sequence, as well as possibly minimalamount of flanking sequences (e.g., 3-30, 6-27, 9-24, 12-21, or 15-18nucleotides), on 5′ and/or 3′ ends, which may be naturally occurringsequences; artificial sequences (e.g., linkers), or combinationsthereof. Such sequences can be present in the context of largersequences (e.g., heterologous virus or other vector sequences).

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the Drawings, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the structural region of CR6 genome (lower panel)and amino acid alignment of NimIV sequences of HRV6 and HRV14 (upperpanel).

FIGS. 2A and 2B are graphs showing the results of plaque reductionneutralization assays of CR6 (a chimera including HRV14 sequences, withthe exception of NimIV sequences, which are HRV6 sequences; alsoreferred to herein as CR6; the right-hand bar of each pair (green)) andHRV14 (the left-hand bar of each pair (brown)) with guinea pigpolyclonal antibodies anti-HRV14 (FIG. 2A) and anti-HRV6 (FIG. 2B). 20K,40K, 60K, 80K correspond to titers of antibodies 2×10⁴, 4×10⁴, 6×10⁴,and 8×10⁴ respectively. The upper (green) and lower (brown) dashed linesindicate 50% reduction of plaque number for HRV14 and HRV6,respectively.

FIGS. 3A-3D are three-dimensional models of HRV14 and CR6. FIGS. 3A and3B are 3D models of an HRV14 virus particle designed on the basis ofknown crystal structure (Che et al., J. Virol. 72:4610-4622 (1998))using Chimera software (http://www.cgl.ucsf.edu/chimera/). VP1, Vp2, andVP2 are shown in dark blue, magenta, and grey colors, respectively. TheHRV14 particle is presented as spacefill model, where Nims arecolor-coded onto its Van-der-Vaals surface. Green, blue, and magentawired surfaces depict NimIII, NimIV, and NimII, respectively. Contact ofNimIV with NimIII is shown to be provided through K287. Note that NimIon this model is covered by NimI-specific Fab17 shown by dark green.

FIGS. 3C and 3D are 3D models prepared using Accelrys Discovery Studiov1.5.1 (Accelrys Software, Inc.). FIG. 3C—Spacefill model of NimI,NimII, NimIII, and NimIV of HRV14 particle. Amino acid residues of Nimsare depicted by Van Der Vaals solid surfaces. Positively and negativelycharged surfaces are shown in blue and red, respectively. FIG.3D—Comparison of spacefill models of HRV14 and CR6 viruses (NimIII andNimIV are only shown). The structure of CR6 was predicted on the basisof known crystal structure (see above) and information on proteinsequence CR6 (see FIG. 1). Note: close contact of positively chargedK287 from NimIV of HRV14 with negative residues of NimIII, whereas inCR6 due to K287T substitution this connection is abrogated.

FIG. 4 shows the results of neutralization of CR6 with mouse anti-HRV37,anti-HRV92, and anti-HRV6 sera. FIG. 4A is an alignment of NimIV forHRV14, HRV37, HRV6, and HRV92. Amino acids are numbered (below)according to an HRV14 template. Identical regions are shown in therectangles (blue). FIG. 4B is a series of graphs showing the results ofplaque reduction neutralization test (PRNT) studies of HRV14 (theleft-hand bar of each pair; brown) and CR6 (the right-had bar of eachpair; green) with anti-HRV37, anti-HRV92, and anti-HRV6 mouse antibodiesgenerated against corresponding purified viruses. 50% neutralizationtiters are shown by either dashed lines on the graphs or numerically(50% NUT) in the boxed panel of the picture beneath correspondentgraphs.

FIG. 5 shows experimental data based on NimIV^(HRV6)- andNimIV^(HRV14)-specific synthetic peptides. FIG. 5A is a Western blot ofKLH-linked peptides H6 (NimIV^(HRV6)) and H14 (NimIV^(HRV14)) detectedby guinea pig anti-HRV14 (GP14) and anti-HRV6 (GP6) polyclonalantibodies. FIG. 5B is a Western blot of free H6 and H14 peptidesdetected with the same antibodies; lane (1)—protein weight marker, lane(2)—H6-KLH (A) or H6 (B), lane (3)—H14-KLH (A) or H14 (B). FIG. 5C is agraph showing the results of ELISA analysis of H6 and H14 with GP6 andGP14.

FIG. 6 is a graph showing the results of plaque reduction neutralizationtest (PRNT) studies of HRV14 and HRV6 with mouse anti-HRV14-NimIV^(HRV6)serum. These data show immunodominance of NimIV^(HRV6) in the backgroundof HRV14 capsid.

FIG. 7 is a graph showing the results of plaque reduction neutralizationtest (PRNT) studies of HRV14 and CR6, which shows that a NimIIImonoclonal antibody (Mab5)) neutralized CR6 about ten fold less thanHRV14.

FIG. 8 is a graph showing the results of plaque reduction neutralizationtest (PRNT) studies of HRV14 and CR6, which shows that a NimIImonoclonal antibody (Mab16) neutralized CR6 about five fold more thanHRV14.

FIG. 9 is a graph showing the results of plaque reduction neutralizationtest (PRNT) studies of HRV14 and CR6, which shows that a NimI monoclonalantibody (Mab17) neutralized CR6 about 1.5 fold less than HRV14.

FIG. 10 is a table showing that Nim IV affects NimI, NimII, and NimIII(50% neutralization titer).

FIG. 11 shows an alignment of NimIII and NimIV sequences, as well as theposition of these sequences in the HRV structural proteins.

FIG. 12A is an alignment of VP1 sequences of CR6 and CR72 chimeras. FIG.12B is a schematic representation of HRV genome, with alignment ofNimIVs of HRV6, HRV72 and HRV14.

FIG. 13 is a pair of graphs showing that NimIV confers unto chimericrecombinant the neutralization characteristics of the donor serotype.FIG. 13A shows neutralization titers of CR72 (open bars) and HRV14(black bars) with GP72 antibodies. FIG. 13B shows neutralization titersof CR6 (open bars) and HRV14 (black bars) with GP6 antibodies. Note: GP6and GP72=guinea pig polyclonal antibodies (ATCC) against HRV6 and HRV72,respectively.

FIG. 14 is a table showing the effect of NimIV replacement on other Nimsof an HRV14 backbone (NimI, II, III Mabs against HRV14, CR6 and CR72(neutralization)).

FIG. 15 is a table showing the 50% neutralization titers of anti-CR6 andanti-CR72 mouse antiserums against HRV14, HRV6, HRV72, CR6, and CR72.

DETAILED DESCRIPTION OF THE INVENTION

In general, the invention relates to a novel immunogenic locus of humanrhinovirus (HRV) and its use in vaccines to prevent or treat HRVinfection. The invention is based on our discovery of a new HRVneutralizing immunogen (Nim), NimIV, which can be used as a vaccine.This vaccine, as described below, comprises several embodiments. Theseinclude single or multiple recombinant HRVs displaying heterologousNimIV antigens, synthetic NimIV peptides alone or in the context ofvirus, protein, or chemically-linked carriers, and mixtures ofbiological or chemical fusions of serotype-diverse NimIV peptides in thecontext of biological carriers. Such HRV vaccines, which elicitNimIV-specific immune responses to a broad array of HRV serotypes, areuseful for both prophylactic and therapeutic treatment of HRV infection.The NimIV antigen, vaccine compositions including NimIV, and methods ofusing such compositions are described further, as follows.

Neutralizing Immunogen IV (NimIV)

Three major surface Neutralizing Immunogens (NimI, NimII, and NimIII) ofrhinoviruses (HRVs) elicit highly specific neutralizing immuneresponses. Nim-specific antibodies block virus attachment to the cellreceptor (ICAM-1). The present invention is based on the discovery of anovel Nim (NimIV), encompassing a stretch of about 17-25 amino-acidsequences at the C-terminal end of structural protein VP1, andidentified by molecular evolution experiments. We demonstrate that NimIVis exchangeable between different HRV serotypes. For example, when NimIVof a donor serotype HRV (e.g., HRV6 or HRV72) is introduced into anotherserotype host virus (e.g., HRV14), it confers on the resulting chimericrecombinant neutralization characteristics of the donor serotype,significantly changing the neutralizing characteristics of the hostvirus. The incorporation of NimIV into recombinant HRV vaccines willresult in serotype cross-reactive immune responses directed against abroad array of HRV serotypes.

Recombinant HRV Vaccine Utilizing Chimeric NimIV Antigens

One characteristic of an ideal HRV vaccine is the ability to protect ahuman at risk of HRV infection from a broad range of HRV serotypes. Thevaccines of the present invention feature the ability to elicitprotective and therapeutic immune responses against a large number ofHRV serotypes (e.g., a majority or, more ideally, all HRV serotypes)that cause disease in humans. This can be accomplished by the use ofmultiple NimIV sequences in a vaccine, which can involve, for example,the addition of NimIV antigens from donor serotypes into a small groupof host serotype HRVs. As we show below, the transferred NimIV antigenprovokes strong neutralizing antibody responses that are serotypespecific. In the context of chimeric or recombinant vaccines, thecombination of a first serotype NimIV antigen into a second serotypehost HRV elicits neutralizing antibodies directed against both HRVserotypes, thus broadening the protective or therapeutic benefit over avaccine not chimeric at the NimIV locus. For example, replacement ofNimIV^(HRV14) (i.e., the NimIV antigen in HRV serotype 14) of HRV14 withNimIV^(HRV6) yields the HRV vaccine CR6 (discussed further below). Thisvaccine induces generation of neutralizing antibodies directed againstboth. HRV14 and HRV6 serotypes. In another example, replacement ofNimIV^(HRV14) of HRV14 with NimIV^(HRV72) yields the HRV vaccine CR72(discussed further below). This vaccine generates neutralizingantibodies directed against both HRV14 and HRV72 serotypes. A mixture ofrecombinant HRVs, thus constructed, that comprise a large number ofdonor serotype NimIV antigens and a limited number of host serotype HRVcombinations represents an ideal vaccine for the prevention or treatmentof HRV infection.

NimIV Peptides

A second embodiment of the invention is the use of synthetic ornaturally-derived NimIV peptides that correspond to the amino acidsequence of the NimIV genetic locus. Examples of such peptides areprovided elsewhere herein (see, e.g., the Summary of the Invention andthe Experimental Examples). The administration of a mixture of peptides,pooled from a broad range of HRV serotypes, elicits a broadly protectiveneutralizing antibody response for the prevention or treatment of HRVinfection. The administration of a mixture of NimIV peptides can occuralone or in combination with pharmaceutically acceptable adjuvants orstimulants of the immune system (see below).

NimIV Fusion Molecules

Another aspect of the invention is the chemical or biological fusion ofNimIV antigens to a biological carrier to be used as an HRV vaccine. Inthis context, NimIV peptides, derived from single or multiple serotypes,are bound to a suitable biological carrier (e.g., a hepatitis B coreantigen) to improve degradation half-life, tissue penetrance andspecificity, detection, or immunogenicity of the NimIV peptides.Mixtures of such NimIV fusion molecules, drawn from many HRV serotypes,are then used to vaccinate a human to prevent or treat HRV infection. Inother examples, NimIV peptides (which may be from many differentserotypes) are cross-linked to HRV carriers.

Administration and Dosage

The present invention also provides compositions that includeprophylactically or therapeutically effective amounts of one or morehuman rhinovirus vaccine, as described herein. The mixtures of HRVvaccines may be present in the same pharmaceutical composition (a singledosage form) or separate pharmaceutical compositions (separate dosageforms), which are administered concomitantly or at different times. Thecompositions can be formulated for use in a variety of drug deliverysystems. One or more physiologically acceptable excipients or carrierscan also be included in the compositions for proper formulation. Theviruses can be in lyophilized form or dissolved in a physiologicallycompatible solution or buffer, such as saline or water. Standard methodsof preparation and formulation can be used as described, for example, inRemington's Pharmaceutical Sciences (18^(th) edition), ed. A. Gennaro,1990, Mack Publishing Company, Easton, Pa.

The compositions are intended for intranasal, parenteral, topical, oral,or local administration for prophylactic and/or therapeutic treatment.Typically, the compositions are administered intranasally (e.g., byaerosol inhalation or nose drops), parenterally (e.g., by intramuscular,subcutaneous, or intravenous injection), or by oral ingestion, or bytopical application or intraarticular injection. Additional routes ofadministration include intravascular, intra-arterial, intratumor,intraperitoneal, intraventricular, intraepidural, as well as ophthalmic,intrascleral, intraorbital, rectal, or topical administration. Sustainedrelease administration is also specifically included in the invention,by such means as depot injections or erodible implants or components.Thus, the invention provides compositions for mucosal or parenteraladministration that include the above-mentioned agents dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier, e.g.,water, buffered water, saline, PBS, and the like. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike. The invention also provides compositions for oral delivery, whichmay contain inert ingredients such as binders or fillers for theformulation of a tablet, a capsule, and the like. Further, thisinvention provides compositions for local administration, which maycontain inert ingredients such as solvents or emulsifiers for theformulation of a cream, an ointment, and the like.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, e.g., between 5 and 9, 6 and 8, or 7 and 8, such as 7 to 7.5.The resulting compositions in solid form may be packaged in multiplesingle dose units, each containing a fixed amount of the above-mentionedagent or agents, such as in a sealed package of tablets or capsules. Thecompositions can also include the active ingredient(s) in lyophilizedform, which is reconstituted for administration.

The compositions containing an effective amount of vaccine can beadministered for prophylactic and/or therapeutic treatments. Inprophylactic applications, compositions can be administered to a subject(e.g., a human subject) with increased susceptibility to HRV infection.Compositions of the invention will be administered to the subject (e.g.,a human) in an amount sufficient to delay, reduce, or prevent the onsetof clinical or subclinical disease. In therapeutic applications,compositions are administered to a patient (e.g., a human) alreadysuffering from HRV infection in an amount sufficient to cure or at leastpartially arrest the symptoms of the condition and its complications. Anamount adequate to accomplish this purpose is defined as a“therapeutically effective dose.” Determination of an appropriate dosageamount and regimen can readily be determined by those of skill in theart. Amounts effective for this use may depend on the severity of thedisease or condition and the weight and general state of the patient,but generally range from about 0.5 mg to about 3000 mg of the agent oragents per dose per patient. The vaccines can be administered one timeonly or in prime/boost regimens. Suitable regimens for initialadministration and booster administrations are typified by an initialadministration followed by repeated doses at one or more hourly, daily,weekly, or monthly intervals by a subsequent administration. The totaleffective amount of an agent present in the compositions of theinvention can be administered to a mammal as a single dose, either as abolus or by infusion over a relatively short period of time, or can beadministered using a fractionated treatment protocol, in which multipledoses are administered over a more prolonged period of time (e.g., adose every 4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, 1-2weeks, once a month).

The therapeutically-effective amount of one or more agents presentwithin the compositions of the invention and used in the methods of thisinvention applied to mammals (e.g., humans) can be determined by thethose of skill in the art with consideration of individual differencesin age, weight, immune system integrity, and the condition of themammal. The agents of the invention are administered to a subject (e.g.a mammal, such as human, mouse, livestock (e.g., cattle, sheep, orpigs), domestic pet (e.g., cat or dog)) in an effective amount, which isan amount that produces a desirable result in a treated subject (e.g.,the prevention of HRV infection in a susceptible individual or thelessening of symptoms in an infected individual). Such therapeuticallyeffective amounts can be determined empirically by those of skill in theart.

The vaccines of the invention can be used in combination with othervaccination approaches, as well as other approaches to treatment (e.g.,small molecule-based approaches). For example, the viruses can beadministered in combination with other recombinant vaccines includingthe same or different antigens. The combination methods of the inventioncan include co-administration of vaccines of the invention with otherforms of the antigen. Alternatively, the vaccines of the presentinvention can be used in combination with other approaches (such assubunit or HBc approaches (HBc-M2e; Fiers et al., Virus Res.103:173-176, 2004; WO 2005/055957; US 2003/0138769 A1; US2004/0146524A1; US 2007/0036826 A1)) in a prime-boost strategy, witheither the vaccines of the invention or the other approaches being usedas the prime, followed by use of the other approach as the boost, or thereverse. Further, the invention includes prime-boost strategiesemploying the vaccine of the present invention as both prime and boostagents.

The vaccines of the invention can be administered to subjects, such asmammals (e.g., human subjects) using standard methods. In the case ofintranasal administration, the vectors can be administered in the formof nose-drops or by inhalation of an aerosolized or nebulizedformulation.

The vectors of the invention can be administered to subjects, such ashumans, as live or killed vaccines. The live vaccines can beadministered intranasally using methods known to those of skill in theart (see, e.g., Grunberg et al., Am. J. Respir. Crit. Car. Med.156:609-616, 1997). Appropriate dosage amounts and regimens can readilybe determined by those of skill in the art. As an example, the doserange can be, e.g., 10³ to 10⁸ pfu per dose. The vaccine canadvantageously be administered in a single dose, however, boosting canbe carried out as well, if determined to be necessary by those skilledin the art. As to inactivated vaccines, the virus can be killed with,e.g., formalin or UV treatment, and administered intranasally at about10⁸ pfu per dose, optionally with appropriate adjuvant (e.g., chitin ormutant LT; see above). In such approaches, it may be advantageous toadminister more than one (e.g., 2-3) dose.

The size of the peptide or protein that is included in a vaccine of theinvention can range in length from, for example, from 3-1000 aminoacids, for example, from 5-500, 10-100, 20-55, 25-45, or 35-40 aminoacids, as can be determined to be appropriate by those of skill in theart. Thus, peptides in the range of 7-25, 12-22, and 15-20 amino acidsin length can be used in the invention. Further, the peptides notedherein can include additional sequences or can be reduced in length,also as can be determined to be appropriate by those skilled in the art.The peptides listed herein can be present in the vectors of theinvention as shown herein, or can be modified by, e.g., substitution ordeletion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more amino acids). In addition, the peptides can be present inthe vaccine in the context of larger peptides. Optionally, peptides suchas those described above and elsewhere herein include additionalsequences on the amino and/or carboxyl terminal ends, whether suchsequences are naturally associated with the peptide sequences (i.e., thesequences with which the peptides are contiguous in the influenza virusgenome) or not (e.g., synthetic linker sequences). The peptides can thusinclude, e.g., 1-25, 2-20, 3-15, 4-10, or 4-8 amino acid sequences onone or both ends. As a specific example, the peptide may include 1-3linker sequences at amino and/or carboxyl terminal ends.

Adjuvants

For vaccine applications, optionally, adjuvants that are known to thoseskilled in the art can be used. Adjuvants are selected based on theroute of administration. In the case of intranasal administration,chitin microparticles (CMP) can be used (Asahi-Ozaki et al., Microbesand Infection 8:2706-2714, 2006; Ozdemir et al., Clinical andExperimental Allergy 36:960-968, 2006; Strong et al., Clinical andExperimental Allergy 32:1794-1800, 2002). Other adjuvants suitable foruse in administration via the mucosal route (e.g., intranasal or oralroutes) include the heat-labile toxin of E. coli (LT) or mutantderivatives thereof. In the case of inactivated virus, parenteraladjuvants can be used including, for example, aluminum compounds (e.g.,an aluminum hydroxide, aluminum phosphate, or aluminum hydroxyphosphatecompound), liposomal formulations, synthetic adjuvants, such as (e.g.,QS21), muramyl dipeptide, monophosphoryl lipid A, or polyphosphazine. Inaddition, genes encoding cytokines that have adjuvant activities can beinserted into the vectors. Thus, genes encoding cytokines, such asGM-CSF, IL-2, IL-12, IL-13, or IL-5, can be inserted together withforeign antigen genes to produce a vaccine that results in enhancedimmune responses, or to modulate immunity directed more specificallytowards cellular, humoral, or mucosal responses. Alternatively,cytokines can be delivered, simultaneously or sequentially, separatelyfrom a recombinant vaccine virus by means that are well known (e.g.,direct inoculation, naked DNA, in a viral vector, etc.).

Experimental Examples Identification of NimIV

We have discovered a neutralizing immunogen, NimIV, which encompasses a17-25 amino acid long, non-conserved sequence of the C-terminus of virusstructural protein 1 (VP 1). This epitope can be exchanged between HRVserotypes. If substituted, NimIV confers its neutralizationcharacteristics to the heterologous HRV. Synthetic peptidescorresponding to NimIV were shown to be recognized by virus-specificantibodies in ELISA and Western blot experiments.

Two viable chimeras HRV14-NimIV^(HRV6) (CR6) and HRV14-NimIV^(HRV72)(CR72) were isolated during a molecular evolution experiment (VP1 geneshuffling) performed as described below. As shown in the alignmentpresented in FIG. 12A, VP1 sequences of CR6 and CR72 included severalindividual amino acid substitutions as well as replacements ofNimIV^(HRV14) to NimIV^(HRV6) and NimIV^(HRV72) in CR6 and CR72respectively. NimIVs alignment (FIG. 12B) showed that all NimIV virusescontain conservative central domain (PVIKKRK/E), while flanking regionswere varied. Interestingly, amino acids at positions 279 and 282 wereshown to be fully conserved or similar within all HRV serotypes(RM2506). CR6 and CR72 chimeras were shown to be strongly neutralizedwith polyclonal guinea pig antibodies GP6 and GP72 (ATCC), while neitherof these antibodies neutralized backbone virus (HRV14; FIG. 13). Mousepolyclonal antibodies derived against HRV6 and HRV72 were also shown toneutralize CR6 and CR72 at 10 fold lower titer then GP6 or GP72.evidenced that NimIV determinants in CR6 and CR72 are surface exposedand in favorable conformation for neutralizing antibody binding.Conformation of these epitopes in chimeras most possibly corresponds tothat in wild type viruses.

DNA Shuffling as a Method of Isolation of NimIV Replacement

Discovery of NimIV was possible after the generation of HRV chimera CR6carrying the replacement of 18 amino acids of the C-terminus part of VP1with the corresponding 17 amino acid region of HRV6 (see FIG. 1). Thissequence was obtained by DNA shuffling (for method review see Patten etal., “Applications of DNA shuffling to pharmaceuticals and vaccines,”Curr Opin Biotechnol 8:724-733 (1997); examples of use include Zhang etal., “Broadly cross-reactive mimotope of hypervariable region 1 ofhepatitis C virus derived from DNA shuffling and screened by phagedisplay library,” J Med Virol 71:511-517 (2003), Castle, et al.,“Discovery and directed evolution of a glyphosate tolerance gene,”Science 304:1151-1154 (2004), Pekrun et al., “Evolution of a humanimmunodeficiency virus type 1 variant with enhanced replication inpig-tailed macaque cells by DNA shuffling,” J Virol 76:2924-2935 (2002),Toth et al., “Improvement of the movement and host range properties of aplant virus vector through DNA shuffling,” Plant J 30:593-600 (2002),Kaper et al., “DNA family shuffling of hyperthermostablebeta-glycosidases,” Biochem J 368:461-470 (2002). Wang et al., “Directedevolution of substrate-optimized GroEL/S chaperonins,” Cell111:1027-1039 (2002), and Hurt et al., “Highly specific zinc fingerproteins obtained by directed domain shuffling and cell-basedselection,” Proc Natl Acad Sci U.S.A. 100:12271-12276 (2003)), followedby cloning this fragment back into HRV14 infectious clone. Approximately100 VP1 sequences were included in the DNA shuffling experiment (Ledfordet al., “VP1 sequencing of all human rhinovirus serotypes: insights intogenus phylogeny and susceptibility to antiviral capsid-bindingcompounds,” J Virol 78:3663-3674 (2004)).

CR6 is Neutralized by Both GP6 and GP14

The neutralization specificity of the CR6 chimera was shown to bedifferent from parental HRV14 vector (pWR3.26 infectious clone). Inaddition to neutralization detected with HRV14-specific polyclonalguinea pig Abs (GP14; FIG. 2A) we found neutralization of CR6 withguinea pig HRV6-specific antibodies (GP6; FIG. 2B), whereas the parentalHRV14 is not neutralized with GP6 (FIG. 2). This indicates that theC-terminus domain of HRV6 is immunogenic and neutralizing.

CR6 is Strongly Neutralized by NimI- and NimII-, but not byNimIII-Specific mAbs

The presence of NimIV^(HRV6) in HRV14 background (CR6) changes NA ofother Nims (HRV14). PRNTs with Nim^(HRV14)-specific mAbs revealed thatCR6 NimIII-specific neutralization was decreased (˜10 fold; FIG. 7),whereas NimII-specific NA was increased (5 fold; FIG. 8); NimI-specificneutralization was only slightly affected (1.5 fold; FIGS. 9 and 16).These findings are summarized in FIG. 10.

Effect of NimIV^(HRV6 and HRV72) on Neutralizing Potency of BackboneNims

To study effect of NimIV replacements on neutralizing characteristics ofbackbone Nims a panel of HRV14 Nim-specific mouse monoclonal antibodieswere used against CR6 and CR72 (FIG. 14). Neutralizing ability of NimIof both chimeras was only slightly if at all affected, whereas NimII andNimIII of CR6 demonstrated 5 fold higher and 10 fold lowerneutralization rates, respectively. In contrast NimIII-dependentneutralization of CR72 was not affected. Unfortunately neutralization ofCR72 with NimII-specific antibodies was not studied since of limit ofantibody supply. These data evidenced for strong interaction betweenNimIV and NimIII domains which are consistent with crystallography andpreviously obtained mutagenesis data.

Modeling of Interactions of NimIV with Other Nims within CR6 and HRV14

These results demonstrate the importance of NimIV HRV6 forconformational integrity of CR6. 3D modeling was performed on the basisof known crystal structure (Che et al., “Antibody-mediatedneutralization of human rhinovirus 14 explored by means of cryoelectronmicroscopy and X-ray crystallography of virus-Fab complexes,” J Virol72:4610-4622 (1998) revealed a close contact of NimIII with NimIV inHRV14, but not in CR6 particles (FIG. 3 B, D). This contact in HRV14 wasassociated with positive charge of K287 of VP1 through which itinteracted with negatively charged residues of NimIII (FIG. 3 B, D). InCR6, mutation to T 287 abrogates this connection (FIG. 3D).Interestingly, the negative effect of mutation at K287 onNimIII-specific neutralization was documented previously (Sherry et al.,“Use of monoclonal antibodies to identify four neutralization immunogenson a common cold picornavirus, human rhinovirus,” J Virol 57:246-2571986)), but the authors claimed that C-terminal region of VP 1 was not aneutralizing immunogen (Nim) due to the absence of escape mutants toneutralization with monoclonal antibodies specific to that region.NimIV^(HRV6) in CR6 only slightly affects NimI-specific neutralization,which could be partially explained by bigger distance of this epitopefrom NimIV (FIG. 3C).

A unique feature of CR6 is its 5 fold higher sensitivity toNimII-specific neutralization (FIG. 14). This enhancement could not beexplained by direct physical contact of NimIV^(HRV6) andNimII^(HRV14. 3)D modeling revealed distant localization of these Nimsin virus particle (FIGS. 3A-C). Most likely this phenomenon could beexplained by conformational changes in VP2, which possibly led to morefavorable to monoclonal antibody binding exposure of NimII on thesurface of virus particle.

Cross-Neutralization Profile of CR6

The alignment of NimIV^(HRV6) with NimIV of all 100 serotypes identifiedits two closest matches: C-terminal ends of HRV37 and HRV92 (see FIG.4A). Analysis revealed the presence of three regions within NimIV:conservative (core) region consisting of 6 AA (P-V-I-K-K-R) and tworegions upstream and downstream from core. Core was also detected inNimIVs of 7 closely related viruses (HRV14, HRV72, HRV83, HRV86, HRV35,HRV79, and HRV3; see FIG. 11). It is worth to note here that R282 wasfound to be conservative among all 100 HRV serotypes. As is shown inFIG. 4A, 6 AA of downstream regions of NimIV^(HRV6) and NimIV^(HRV37)are almost identical (D/E-N-I-T-T-Y), whereas corresponding sequence ofHRV92 is quite different (S-L-I-T-N-Y) from them. Upstream regions ofNimIV^(HRV6) and NimIV^(HRV92) have two identical amino acids, whereasthe corresponding region of NimIV^(HRV37) exposes no apparent similaritywith NimIV^(HRV6). This difference between NimIVs provided anopportunity to assess which portion of the epitope is important forneutralization of CR6 virus. To study this we generated mouseconvalescent sera against all three serotypes and tested them forneutralization of CR6 (FIG. 4B). In spite of extensive homology betweendownstream regions of NimIV^(HRV6) and NimIV^(HRV37) anti-HRV37, serarevealed no neutralization, confirming the insignificance of thedownstream region for neutralization. Conversely, anti-HRV92 serademonstrated only slightly decreased NA than anti-HRV6. None of thesethree sera samples was able to neutralize HRV14. These results representa functional dissection of NimIV, providing evidence for highercross-neutralization activity of upstream versus core and downstreamregions. To answer the question of whether differential recognition ofthese viruses by mouse antibodies reflects their real interaction withNimIV-specific sequences, we synthesized NimIV^(HRV14) andNimIV^(HRV6)-specific peptides and performed Western and ELISA assayswith the same set of antibodies.

Immunoreactivity of NimIV-Specific Peptides GP14 and GP6 DifferentiateBetween Serotype Specific Peptides

Gp6 and GP14 recognize specifically homologous NimIV-specific peptidesin Western blot (FIG. 5A-B) and ELISA (FIG. 5C) assays. FIGS. 5A and 5Brepresent Western blot results with KLH-bound materials and freepeptides respectively. Due to the high molecular weight of KLH (˜3×10⁵kDa) the protein bands on FIG. 5A appear smeared. The immunoreactivityof given peptides are very specific since no signals were detected withheterologous combinations of peptide/antibody (GP6/NimIV^(HRV14) orGP14/NimIV^(HRV6)). Traces of signal in heterologous combinations inKLH-bound material are attributed to features of KLH. These results areevidencing about linearity and high specificity of NimIV epitopes on thesurface of HRV6 and HRV14 purified samples of which were used forgenerating GP6 and GP14, respectively. No apparent cross-reactivitybetween these peptides witnessed about low immunogenicity of core partof these Nims. If this statement were not true, highcross-immunoreactivity should be seen in this experiment.

High specificity of recognition of these peptides with GP6 and GP14 isalso confirmed by ELISA (FIG. 5C). Lower reactivity of H14 with GP14,then H6 with GP6 could indicate on the difference in NimIV epitopepresentation on the surfaces of virus particles. These results arereciprocal to PRNT data described in FIG. 2. In both experiments noapparent cross-reactivity between HRV14 and HRV6 or their NimIV-specificpeptides was identified.

In Vivo Studies: Anti-CR6 Serum Neutralizes HRV6

11-12 week old female Blb/c mice were immunized three times (on days 1,14, and 28) intraperitoneally with either virus suspensions (10⁵ pfu/ml)mixed with adjuvant (aluminum hydroxide), or mock (diluent), in a 100 μlvolume. Mice were terminally bled on day 49. To test for serum antibodylevels, mice were bled prior to inoculation (baseline) and on day 30-40after immunization via the retro-orbital route under isofluoraneinhalation anesthesia or via mandibular route without anesthesia (volumeno more than 7.7 μl/g body weight). PRNT assay demonstrated specificneutralization of HRV6 with the serum pool from 2 mice (FIG. 6). It alsoshowed decreased neutralization of HRV14 virus, which provides evidencethat NimIV^(HRV6) in CR6 is the immunodominant epitope.

Methods Peptides and Conjugates

Oligopeptides NimIV^(HRV6), Nim^(HRV72), and NimIV^(HRV14) correspondingto C-terminal ends of structural regions of HRV6 (CKNIVPVIKKRENITTY),HRV14 (CNTEPVIKKRKGDIKSY) and HRV72 (CNPKPVIKKREGDIKTY) respectivelywere prepared by standard solid-phase synthesis by Biosynthesis, Inc(Lewisville, Tex.). Part of peptide materials were conjugated to aHemocyanin from Concholepas concholepas (KLH) by use of crosslinkersuccinimidyl-4-(p-maleimidophenyl)-butyrate (sMBS) and reducing agentTCEP.HCl Tris(2-carboxyethyl)phosphine hydrochloride (TCEP HCL).

Cell Culture, Viral Propagation and Reagents

HRV serotypes 6, 14, 35, 37, 72, 83, 86, 92 stocks (ATCC) were amplifiedto high titer by successive infection of target H1 HeLa cells. HeLacells (ATCC) were maintained in Minimum Essential Medium (Invitrogen)with 5% fetal bovine serum (JRH Biosciences, KS) for routinepropagation. Cells were maintained under subconfluent growth conditionsduring passage. After 48 hours at 34° C., viruses were released from thecells by three freeze-thaw cycles at −80 and 37° C. The cell debris wasdiscarded, while supernatant containing amplified virus was aliquotedand frozen at −80° C. Guinea pig antiserum for HRV serotypes 6, 14, 72,92, and 37 were obtained from the ATCC.

VP1 Gene Shuffling Virus Libraries

DNA fragments of VP1 are amplified by RT-PCR from RNA of HRV serotypes6, 14, 35, 37, 72, 79, 83, 86, and 92. For the purpose of furthercloning internal AvrII sites presented in VP1 genes of HRV serotypes 83,86, 92 are removed by virtue of recombinant PCR. All PCR fragments arepooled together and shuffled, followed by cloning in modified HRV14 cDNAvector pWR3.26 (ATCC). Briefly, two microgram of pooled PCR fragmentsare treated with DNase I (Amersham Pharmacia Biotech, Inc) and afraction of 50-100 bp DNA fragments is gel purified and subjected to15-25 cycles of PCR without primers at 94° C. 30 sec, 50° C. 1 min, 72°C. 1 min followed by 25 cycles PCR with cloning primers at 94° C. 30sec, 55° C. 30 sec, 72° C. 1 min. Library of amplified shuffled VP1sequence are cloned into the modified pWR3.26 plasmid at XhoI and AvrIIsite. For that purpose HRV14 cDNA clone pWR3.26 is modified by insertingXhoI site at 5′ site of VP1 sequence (FIG. 12). XhoI and AvrII sites areincorporated into VP1 forward and reverse cloning primers respectively.

VP1 shuffling plasmid DNA library is linearized by MluI digestion andtranscribed in vitro by T7 transcription kit (Epicentere, Inc). RNA istransfected into H1-Hela cell (ATCC) by lipofectine (Invitrogen, Inc).Cells are harvested after incubation at 34° C. for 2-4 days. Cellsamples are subjected to three freeze-thaw cycles and the supernatant isused to infect monolayer of H1-Hela cells. Virus library are stored at−80° C.

Isolation of HRV14-NimIV Recombinant Viruses

HRV14-NimIV^(HRV6) (CR6) chimera is plaque purified from virus librarydescribed above. To isolate other HRV14-NimIV^(HRVX) recombinants totalRNA from virus library is used as a template for 8 different RT-PCRreactions performed with 8 serotype-specific reverse primers annealingto 3′-ends of VP1 gene. The same forward primer complimentary toconservative region upstream to VP1 gene was used in all of thesereactions. Resulting PCR fragments are cloned back into pWR3.26 plasmidas described above for VP1 shuffliants. After transcription andtransfection into H1 Hela cells, individual viruses are plaque purifiedand sequenced.

Animal Protocols

8 week old female Balb/c mice (10 mice per group) are primed on day 0,then boosted on days 14 and 28 by intraperitoneal administration offiltered cell culture medium containing ˜1.0×10⁶ pfu per dose of either(1) HRV14-NimIV^(HRV6), (2) HRV14-NimIV^(HRV72), (3) parental HRV14, ormock (culture supernatant) as a negative control, mixed with 100 μg ofadjuvant (aluminum hydroxide) in a 500 μL volume.

NimIV^(HRV6) and NimIV^(HRV6), coupled (or not) to KLH peptides are usedfor immunization of 8 week old female Balb/c mice. Mice are primed onday 0 with 100 μl of 15 μg of KLH-bound peptide in Titermax Gold (1:1emulsion) via the subcutaneous route and boosted twice (on day 36 andday 49) by intraperitoneal administration of 15 μg of “free” peptidesdissolved in 100 μl of PBS.

NimIV-specific antibody titers in sera are determined by an establishedELISA performed in microtiter plates coated with corresponding syntheticNimIV peptides.

Plaque Reduction Neutralization Test (PRNT)

Approximately 50 pfu of studied HRV (in complete MEM+5% FBS culturemedium) is mixed with various dilutions of sample serum in a totalvolume of 3004 and incubated overnight at 4° C. One hundred microlitersof each mixture is used to infect one well of H1 Hela cells in a 12wells tissue culture plate (seeded at 6×10⁵ H1-HeLa cells per well andincubated overnight in a 37° C. incubator). After 1 h incubation at 34°C., the cells are overlaid with 1 mL of 0.4% agarose in MEM, 10% FBSwith Pen/Strep and incubated at 34° C. for approximately 3 days. Themonolayers are then fixed with formaldehyde (3.7% final concentration)and stained with 1% crystal violet in 70% methanol.

ELISA

96 well plates are coated with 5 μg/ml of NimIV-specific peptides orpurified HRV14 virus for overnight at 4° C. Plates are incubated withantiserum in different dilutions for 1 hr at 37° C. followed with 1:1000goat anti-mouse IgG-AP conjugated (Southern Biotech, Inc) for 1 hour at37° C. Plates are developed in alkaline phosphatase substrate asdescribed by vendor (Sigma, Inc).

Western Blot

20 μg peptide are loaded on 10% tris-glycine SDS gel (Novex, Invitrogen,Inc) after a short time of electrophoresis running, peptide istransferred onto nitrocellulose membrane (Bio-Rad, Inc). Non-specificbinding to membrane is achieved by soaking membrane in blocking solution(5% non-fat milk in PBS/0.05% tween) for 1 hr at room temperature.Membranes are incubated with guinea pig anti-HRV6 or anti-HRV 14polyclonal antibodies (ATCC) at 1:1000 in blocking solution forovernight at 4° C. After three 15 minute washes in PBS/0.05% Tween,membranes are incubated with goat anti-mouse IgG-AP conjugated antibody(Southern Biotech) in blocking solution for 1 hr at room temperature.Membrane was developed in AP substrate (Sigma SIGMA FAST™ BCIP/NBT) for10 minutes.

Other Embodiments

All publications, patent applications, and patents mentioned in thisspecification are incorporated herein by reference.

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific desiredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments.

Indeed, various modifications of the described modes for carrying outthe invention that are obvious to those skilled in the fields ofmedicine, pharmacology, or related fields are intended to be within thescope of the invention. Use of singular forms herein, such as “a” and“the,” does not exclude indication of the corresponding plural form,unless the context indicates to the contrary.

What is claimed is:
 1. An isolated rhinovirus neutralizing immunogen IV(NimIV) peptide.
 2. The peptide of claim 1, wherein the NimIV peptide isa human rhinovirus 14 (HRV14) NimIV peptide.
 3. The peptide of claim 1,wherein the NimIV peptide comprises amino acids 277-283 of the carboxylterminal region of virus structural protein 1 (VP1) of a humanrhinovirus.
 4. The peptide of claim 3, wherein the peptide comprisesamino acids 275-285 of the carboxyl terminal region of VP1 of a humanrhinovirus.
 5. The peptide of claim 1, wherein the sequence of saidpeptide comprises a sequence selected from the group consisting of:PVIKKR, PVIKKRK (HRV14), PVIKKRE (HRV6 and HRV72), PVIKKRS (HRV92),PVIEKRT (HRV83), PKIIKKR (HRV86), PVIKRRE (HRV35), PIIAKRE (HRV79),TIIKKRT (HRV3), NTEPVIKKRKGDIKSY (HRV14), and A-X₁-X₂-I-X₃-X₄-R-X₅-B,where X₁=P or T; X₂=V, K, or I; X₃=K, E, I, or A; X₄=K or R; X_(s)=S, E,D, T, R, T, or K; A=0-10 additional amino acids; and B=0-10 additionalamino acids.
 6. An isolated nucleic acid molecule encoding a NimIVpeptide or the complement thereof.
 7. A vector comprising an isolatedNimIV peptide or nucleic acid molecule.
 8. The vector of claim 7,wherein the vector is a human rhinovirus vector.
 9. The vector of claim8, wherein the human rhinovirus vector is of a serotype different fromthat of the human rhinovirus from which the NimIV peptide is derived.10. The vector of claim 9, wherein the NimIV peptide or nucleic acidmolecule is present in said human rhinovirus vector in place of NimIVsequences originally present in said vector.
 11. The vector of claim 8,wherein the human rhinovirus vector is a human rhinovirus 14 (HRV14)vector.
 12. The vector of claim 7, wherein the human rhinovirus fromwhich the NimIV peptide is derived is human rhinovirus 6 (HRV6) or humanrhinovirus 72 (HRV72).
 13. The vector of claim 8, wherein the humanrhinovirus vector is a human rhinovirus 14 (HRV14) vector and said humanrhinovirus from which the NimIV peptide is derived is human rhinovirus 6(HRV6) or human rhinovirus 72 (HRV72).
 14. The vector of claim 10,wherein the VP1 protein or nucleic acid molecule of said vector isreplaced with the VP1 protein or nucleic acid of the human rhinovirusfrom which the NimIV peptide is derived.
 15. The vector of claim 7,wherein the vector comprises an inactivated human rhinovirus, to whichthe NimIV peptide is cross-linked.
 16. The vector of claim 7, whereinthe vector comprises a hepatitis B core sequence to which NimIVsequences are fused.
 17. A pharmaceutical composition comprising thepeptide of claim 1 or the nucleic acid molecule of claim
 6. 18. Thepharmaceutical composition of claim 17, wherein the peptide is comprisedwithin a vector.
 19. The pharmaceutical composition of claim 17, furthercomprising one or more of a pharmaceutically acceptable diluent,excipient, carrier, or adjuvant.
 20. The pharmaceutical composition ofclaim 19, wherein the adjuvant is selected from the group consisting ofa chitin microparticle and an aluminum compound.
 21. The pharmaceuticalcomposition of claim 17, further comprising one or more additional humanrhinovirus neutralizing immunogens.
 22. A method of inducing an immuneresponse to a rhinovirus in a subject, the method comprisingadministering to the subject an isolated NimIV peptide or nucleic acidmolecule.
 23. The method of claim 22, wherein the subject does not havebut is at risk of developing rhinovirus infection.
 24. The method ofclaim 22, wherein the subject has rhinovirus infection.